Plastproduktionen och användningen av plast ökar varje år i hela världen och detta är ett av världens största problem. Plaster är ett svårnedbrytbart ämne och processen kan ta hundratals år. Detta leder i sin tur till att plastackumuleringen orsakar skadlig påverkan på klimat, miljö och människor. Ett av alternativen som har stor potential för hanteringen av detta problem är att minska användningen av plast och i stället öka produktionen och användningen av bioplastereller biopolymerer som är nedbrytbar. Bioplaster har kortare nedbrytningstid än vanliga plaster och därför är bioplast ett bra alternativ istället för att använda vanliga plaster. Det finns tre olika grupper av bioplaster, nämligen dem som är biobaserade, bionedbrytbara och de som är både biobaserade och bionedbrytbara. Entyp av bioplast som är biologiskt nedbrytbar och biobaserad är polyhydroxialkanoater (PHA)som används i olika branscher då PHA är en av de biopolymerer som visar störst potential attersätta plast i framtiden. Forskning pågår på Högskolan i Borås med målet av att bilda en ny process för produktion och återvinning av PHA. Processen baseras på produktion av PHA från flyktiga fettsyror som produceras från acidogen jäsning. Acidogen jäsning är en modifierad process av anaerobmatsmältning som används idag för produktion av biogas från avfall. Den kan vara den rättaprocessen för återvinningen av PHA-baserade avfall och för att producera ett billigt substrat, nämligen flyktiga fettsyror för PHA produktion. Det här projektet handlar om en del av processen, nämligen produktion av PHA med bakterier. Forskningsgruppen på Högskolan iBorås har en bakteriestam som inte har undersökts för produktion av PHA. Innan bakterien kan studeras för produktion av PHA från flyktiga syror behövs medelinnehållet optimeras för bakterietillväxt. Syftet med detta arbete är att undersöka effekten av olika glukoskoncentrationer som kollkälla och olika mängder av ammoniumsulfat som används vid tillväxt av den bakterien Bacillusmegaterium för produktion av PHA. En jämförelse mellan det definierade mediet som användsi detta arbete och dess ersättning med nutrient broth, ett komplext medium som normalt används för bakterietillväxt genomfördes också för att utvärdera lämpligheten hos mediet underutveckling för att framgångsrikt stödja bakterietillväxt. Under detta arbete användes flera olika analytiska tekniker såsom pH mättning, Högupplösande vätskekromatografi (HPLC),Spektrometer (OD mättning), och Fourier-transform infraröd spektroskopi (FTIR) för att utvärdera bakterietillväxt och produktion av PHA samt analysera sammansättningen av PHAs. Mängden av ammoniumsulfat påverkade glukosförbrukningen, där koncentrationer av 3 g/Loch 7 g/L visades att leda till en snabbare glukosförbrukning jämfört med 5 g/L. Därför valdes3 g/L ammoniumsulfat då det innehåller mindre kemikalier användning och påverkar inte celltillväxten på ett negativ sätt. Under experimentet med användning av 3 g/Lammoniumsulfat med 10 g/L glukos, konsumerades glukosen fullständigt vid 72 timmar av bakterietillväxt och den maximala PHA-produktionen var på 13–14% baserad på torr cell vikt. Tillväxtmedelet som utvecklades i det här projektet visade att vara lämplig för bakterietillväxt eftersom användning av nutrient broth som används normalt för bakterietillväxt ledde till långsammare glukosförbrukning. Initial glukoskoncentration (5, 10 och 20 g/L) påverkade inte glukosförbrukningen och får studerats vidare för att öka cellkoncentrationen och följaktligen produktionen av PHA. En av det mest studerade i PHA-familjen är polyhydroxibutyrat (PHB). Under detta arbete visade det sig att genom användning av FTIR att PHB framställdes. / Plastic production and use of plastic are increasing every year throughout the world and this is one of the world's biggest problems. Plastic is a persistent substance, and its biodegradation process can take hundreds of years. This in turn leads to the accumulation of plastic causing harmful effects on the climate, the environment, and people. One of the alternatives that has great potential when it comes to dealing with this problem is to reduce the use of plastics and instead increase the production and use of bioplastics or biopolymers that are biodegradable. Bioplastics have a shorter biodegradation time than plastics and therefore bioplastics are a good alternative instead of using ordinary plastic. There are three different groups of bioplastics, namely those that are bio-based, those that are biodegradable and those that are both bio-based and biodegradable. One type of bioplastic that is biodegradable and bio-based is Polyhydroxyalkanoates (PHA) which are used in various industries as PHA is one of the biopolymers that shows the greatest potential to replace plastic in the future. Research is underway at the University of Borås with the goal of developing a new process for the production and recycling of PHAs. The process is based on the production of PHAs from volatile fatty acids produced from acidogenic fermentation. Acid fermentation is a modified process of anaerobic digestion; the latter is used nowadays to produce biogas from waste and can be the right process for recycling PHA-based waste and for producing a cheap substrate, namely volatile fatty acids for PHA's production. This project is about a part of the process, namely the production of PHAs with bacteria. The research group at the University of Borås has a bacterial strain that has not been yet investigated for the production of PHAs. Before this bacterium can be studied for the production of PHAs from volatile fatty acids the compositionoptimization of the cultivation medium for bacterial growth is needed. The purpose of this work is to investigate the effect of different glucose concentrations as a source of carbon and different amounts of ammonium sulphate in the growth of the bacterium Bacillus megaterium and the production of polyhydroxyalkanoates. A comparison between the defined medium under development in this project with nutrient broth, a complex medium normally used to grow bacteria, was also carried out in order to evaluate the suitability of the defined medium to support bacterial growth. During this work several different analytical techniques have been used such as pH measurement, High-Performance Liquid Chromatography (HPLC), Spectrometer (for optical density (OD) measurement), and Fourier-Transform Infrared Spectroscopy (FTIR) to evaluate bacterial growth and production of PHA as well as PHAs composition. The amount of ammonium sulphate affected glucose consumption rate, where concentrations of 3 g/L and 7 g/L were shown to lead to a faster glucose consumption compared to that at 5 g/L. Therefore, 3 g/L ammonium sulphate was chosen as it represents less chemical consumption while not affecting cell growth negatively. During bacterial cultivation in a medium containing 3 g/L ammonium sulphate, 10 g/L glucose, among other compounds, glucose was completely consumed after 72 hours of bacterial growth and the maximum PHA production was 13-14% based on cell dry weight. The cultivation medium developed in this project was shown to be suitable for bacterial growth since the use of nutrient broth, normally used for bacterial growth, led to slower glucose consumption. Initial glucose concentration (5, 10 and 20 g/L) did not affect glucose consumption rate and should be further studied to increase cell concentration and consequently the production of PHA. One of the most studied polymers in the PHA family is polyhydroxybutyrate (PHB). During this work, it was found, through the use of FTIR, that PHB was produced.

This diploma thesis focuses on an optimalization of simple laboratory model systems which serve as an innovative tool for an experimental study on the adsorption of bacterial cells on solid surfaces. In the description of living biological systems, an adsorption is labelled as an adhesion. Designed model systems were validated with a physical-chemical analysis. Various techniques were used to determine bacteria properties, more specifically Burkholderia cepacia and Bacillus megaterium. The solid surfaces after sorption of bacterial cells of Bacillus megaterium were subjected to a structural and visual analysis. Applying the theoretical approach (e.g. using different physical-chemical models) to study the adhesion of microorganisms to a particular surface allows a prediction of the conditions for a successful adhesion. The results will give us a better understanding of a formation and development of a biofilm.

This diploma thesis deals with the change of production characteristics of model plants after application of vermicompost with addition of bacterial inoculum. In this work is elaborated a literature review focused on the process of vermicomposting, composting and stimulation of the root system of cultural crops using symbiotic or growth-promoting microorganisms. In addition, a practical experiment was carried out - an in-house vessel experiment with model plants was established, which was evaluated. The resulting data suggest that the most suitable variants in the formation of above-ground biomass were those containing vermicompost. The highest weight of aboveground biomass was achieved in vermicompost with the addition of Rizocore, where the production of aboveground biomass was increased 1.5 times compared to control soil.

Les catalyseurs chimiques sont utilisés dans différents procédés de synthèse. Cependant, la pollution qu'ils causent sur l'environnement n’est pas prise en considération. Les procédés de synthèse chimique nécessitent généralement un grand volume de solvants organiques, produisant d’énormes quantités de déchets chimiques, souvent toxiques et non dégradables. Le remplacement des catalyseurs chimiques par des biocatalyseurs (enzymes) pourrait donc bénéficier de leur nature écologique et de leur grande sélectivité envers les produits désirés. Néanmoins, la faible activité et stabilité des enzymes ainsi que leurs coûts élevés sont des obstacles majeurs au développement des systèmes enzymatiques. Par conséquent, des études axées sur le développement de systèmes biocatalytiques plus actifs, stables et rentables, pouvant ouvrir les portes vers un environnement plus vert, sont très souhaitables. Parmi les enzymes qui catalysent des réactions d’importance dans de nombreux procédés de synthèse, le cytochrome P450 BM3 issu de Bacillus megaterium fait l'objet de cette thèse. L'enzyme est capable d’hydroxyler les liaisons C–H des acides gras (C₁₂-C₂) à température ambiante et pH physiologique. Pour cette réaction, BM3 n'a besoin que d’oxygène et de deux électrons habituellement obtenus de son cofacteur naturel, le NADPH. Cependant, pour engager cette enzyme dans les réactions d'hydroxylation, quelques obstacles importants doivent être surmontés : (i) le cofacteur coûteux (NADPH), devrait être remplacé par une source d'électrons moins chère ou régénérée, (ii) la stabilité enzymatique devrait être améliorée et (iii) l'enzyme devrait être facilement récupérable du milieu de réaction pour être réutilisée. Dans ce contexte, cette étude propose pour la première fois l'immobilisation d'un BM3 sur des nanoparticules magnétiques (NMP) d’oxyde de fer. Ce système enzymatique bénéficie (i) de la préférence de l'enzyme pour les cofacteurs NADH et BNAH (moins chers que le NADPH), (ii) de la réutilisation facile du biocatalyseur et (iii) d’une stabilité significative de l’enzyme lors du stockage. Les NMP synthétisées ont été fonctionnalisées pour permettre l’immobilisation de l'enzyme par adsorption ou liaison covalente. Par conséquent, les BM3-NMP adsorbées / réticulées ou liées de façon covalente ont été obtenues en immobilisant P450 BM3 (R966D / W1046S) sur Ni²⁺-PMIDA-NMP ou sur des NMP activés par glutaraldéhyde, respectivement. / L'activité de l’enzyme immobilisée a été comparée avec celle de l’enzyme libre dans la réaction d'hydroxylation du 10-pNCA comme substrat modèle. L'acide myristique a également été utilisé comme substrat modèle pour confirmer la capacité d’hydroxylation sélective de l’enzyme sur les atomes de carbone ω-1, -2 ou -3. Pour les mêmes conditions opératoires, le BM3 adsorbé / réticulé a montré plus de 85% de l'activité de l’enzyme libre, alors que pour les BM3-NMP liées de manière covalente cela représente 60%. La séparation facile des NMP du milieu réactionnel à l’aide d’un aimant a permis de réutiliser le système enzymatique cinq fois consécutives. Après 5 cycles de réaction, l'enzyme réticulée a conservé 100% de son activité initiale. Compte tenu que le recyclage de l’enzyme libre n’est pas faisable, ce résultat est d’une importance considérable dans les applications pratiques. De plus, la stabilité de l’enzyme pendant un mois de stockage à 4 ºC a été évaluée pour chaque système de BM3. Les résultats ont montré que l’enzyme libre n’était plus active après seulement une semaine de stockage dans ces conditions. L'enzyme réticulée n'a montré qu'une activité relative de 41% après un mois de stockage, mais pour le BM3 fixée de façon covalente, la valeur correspondante a été de 80%. La cinétique de l'hydroxylation du 10-pNCA en présence de l’enzyme libre ou immobilisée a été également étudiée. Sur la base des données expérimentales, un modèle de Hill (coefficient de Hill égal à 2) a été obtenu pour l'enzyme libre. Il a été démontré que les mêmes paramètres cinétiques sont capables de prédire le comportement du système BM3-adsorbé et BM3-réticulé dans la réaction d’hydroxylation, étant donné sa similarité avec celui de l’enzyme libre. En conclusion, les résultats de cette thèse ont montré qu'un système enzymatique actif, stable et rentable peut être obtenu en immobilisant le BM3 sur des NMP fonctionnalisées. Il bénéficie autant des avantages de l'enzyme que du support. Ainsi, l'immobilisation sur des NMP d’une enzyme spécialement conçue pour remplacer le couteux NADPH par des cofacteurs moins chers mais efficaces (NADH et BNAH) offre en même temps une amélioration significative de sa stabilité et facilite son recyclage. / MNPs have been synthesized and surface functionalized to attach the enzyme via two different methods, adsorption and covalent binding. Moreover, glutaraldehyde was used to treat the adsorbed enzyme molecules on MNPs (crosslinking-adsorption). Therefore, adsorbed, crosslinked-adsorbed, or covalently bound BM3-MNPs were obtained by immobilizing P450 BM3 on synthesized Ni²⁺-functionalized MNPs or glutaraldehyde pre-activated MNPs, respectively. The immobilized enzyme activity was compared to its free counterpart in hydroxylation reaction of 10-pNCA (10-(4-Nitrophenoxy) decanoic acid) as a substrate model. Myristic acid was also used as a substrate model to confirm the enzyme selective hydroxylation at ω-1, -2, or -3 carbon positions. The effect of cofactor (NADH and its analogue, BNAH) on the enzyme activity was also investigated. The adsorbed/crosslinked-adsorbed BM3 showed more than 85% of the free enzyme activity while the covalently bound BM3-MNPs presented 60% of the free enzyme activity under the same reaction conditions. An important feature of BM3-MNPs system is the possibility of recycling the biocatalyst. Facile separation of the magnetic nanoparticles from the reaction medium by applying a magnet provided the opportunity of reusing the enzymatic system for five times. After 5 cycles of reaction, the crosslinked-adsorbed enzyme retained 100% of its initial activity. Although the covalently bound enzyme showed, only half of the crosslinked-adsorbed enzyme activity, its storage stability was more significant. Taking into account that the enzyme reuse is an essential concern in many large-scale applications and the free BM3 cannot be recovered and reused, this result is noteworthy. Storage stability tests revealed that the free enzyme became inactive after one-week while the crosslinked-adsorbed enzyme and the covalently attached BM3 on MNPs showed 41% and 80% relative activity after one month, respectively. Finally, the steady-state kinetics of 10-pNCA hydroxylation by free and immobilized BM3 was investigated. Based on the experimental data, a non-Michaelis-Menten, Hill model (Hill coefficient of 2) was obtained for the free enzyme which could also predict the adsorbed and crosslinked-adsorbed BM3-MNPs system performance. This sigmoidal behavior was found to be independent of enzyme concentration and type of cofactor. However, since the enzyme activity was only 60% of the free enzyme for covalently bound BM3, further studies are necessary for a better understanding of this system. In summary, the results of this thesis show that an active, stable, and cost-effective BM3-MNPs system can be obtained by immobilizing an engineered BM3 on functionalized MNPs. Such systems benefit from the advantages of both enzyme and support. An engineered enzyme can fulfill the desired targets including the replacement of costly NADPH by less-expensive, yet effective cofactors namely NADH and BNAH. Furthermore, immobilization of this enzyme on MNPs improves its stability and facilitates the recycling process. / Chemical catalysts are used in different synthetic processes from lab to industrial scales. High reaction yields usually achieved by this type of processes favor their application in many industries without considering the pollution they cause to the environment. Chemical synthesis processes usually require a high volume of organic solvents and produce tons of chemical wastes which are often toxic and not degradable. Replacing conventional catalysts by biocatalysts (enzymes) can benefit from their environmentally friendly nature and high selectivity toward the desired products. Although the advantages of biocatalysts over chemical catalysts have been proven, the application of enzymes in an industrial level is still not considerable. The enzyme low activity, stability, and high cost are the main concerns in developing large-scale enzymatic systems. Therefore, in the context of a greener environment, studies focusing on the development of more active, stable, and cost-effective enzymatic systems are in great demand. Among several enzymes that can catalyze essential synthesis reactions, cytochrome P450 BM3 from Bacillus megaterium is the subject of this thesis. This enzyme hydroxylates the saturated and unsaturated C–H bonds of medium to long chain fatty acids at room temperature and physiological pH. For this reaction, BM3 only needs molecular oxygen and two electrons usually obtained from its natural cofactor, NADPH. However, to engage this enzyme in hydroxylation reactions, some important obstacles should be overcome: (i) the costly cofactor (NADPH) should be replaced by a cheaper source of electrons or regenerated, (ii) the enzyme stability should be improved, and (iii) the enzyme should be easily recovered from the reaction medium to be reused. In this context, this study proposes for the first time the immobilization of an optimized BM3 mutant on functionalized iron oxide magnetic nanoparticles (MNPs). This enzymatic system benefits from (i) the enzyme preference towards cofactors like the reasonably priced NADH and the very cheap BNAH, (ii) facile recovery and reuse of the biocatalyst (enzyme-MNPs), and (iii) the enzyme significant storage stability.

Au cours de ce travail de thèse intitulé " Identification chimique de métabolites secondaires de certains microorganismes, évaluation de leurs effets dans les domaines pharmaceutiques et agronomiques ", nous nous sommes intéressés à l'étude des effets des conditions de culture sur la production des composés organiques volatils microbiens à partir de deux souches bactériennes co-existantes dans le sol Français : Burkholderia sp. et Bacillus megaterium. A partir des différents extraits préparés, plus que cent composés ont été identifiés, comprenant les dicétopipérazines, les alcools, les composés soufrés, les esters et les acides carboxyliques, par le biais de plusieurs techniques chimiques, analytiques et spectroscopiques. Les résultats obtenus ont montré que les conditions de culture sont les pricipales responsables de la production des différentes familles chimiques des volatiles. Nous avons identifiés des composés qui sont rapportés pour la première fois à partir des bactéries tel que: la N-butylbenzènesulfonamide, triacontane, le proponaoate de 3- (3,5-di-tert-butyl-4-hydroxyphényl), (E) -5-chloro-3-(hydroxyimino) indoline-2-one et 1,3,5-triméthyl-2-octadecylcyclohexane. Sur le plan biologique, on a montré que les résultats obtenus sont fortement influencés par les conditions de culture utilisées pour cultiver les bactéries testées. En parallèle à cette investigation, nous avons montré que les extraits de Burkholderia sp. sont dotés d'un très important potentiel allélopathique. Enfin, une série des analogues de dicétopipérazines a été préparée et évaluée pour leurs activités anti-xanthine oxydase, anti a-amylase et anti 5-lipoxygénase ainsi que pour leurs activités cytotoxiques contre les lignées cellulaires suivantes ; OVCAR, MCF7 et HCT116. Un certain nombre de ces dérivés de dicétopiperazine ont montré des activités anti a-amylase et cytotoxique importantes. / In this thesis entitled " Chemical identification of secondary metabolites from microorganism, evaluation of their effects on pharmaceutical and agronomic fields ", we are interested in studying the effect of culture conditions on the production of microbial volatiles organic compounds by two bacteria that inhabit French soil which are: Burkholderia sp. and Bacillus megaterium. From different prepared extracts, more than one hundred compounds were identified, including diketopiperazine, alcohols, sulfur containing compounds, esters and carboxylic acids, by means of several chemical, analytical and spectroscopic techniques. Results showed that culture conditions of different bacteria are the mainly responsible of production of different blend of volatiles. Many identified compounds including N-butylbenzenesulfonamide, triacontane, octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, (E)-5-chloro-3-(hydroxyimino)indolin-2-one and 1,3,5-trimethyl-2-octadecylcyclohexane are reported for the first time from bacteria.Biologically, we have shown that obtained results are greatly influenced by the cultures conditions used in cultivation of tested bacteria. In addition to that, we have shown that Burkholderia sp. extracts possessed a very good allelopathic potential. Finally, a series new protested deketopiperazine derivatives have been prepared and evaluated in vitro against xanthine oxidase, a-amylase and 5-lipoxygenase enzymes, OVCAR, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of a-amylase and different cancer cell lines.

Made available in DSpace on 2016-06-02T19:55:28Z (GMT). No. of bitstreams: 1 DoutLMP.pdf: 2279571 bytes, checksum: 007958befffa499c1eb6c72cc2b515c8 (MD5) Previous issue date: 2003-06-26 / Universidade Federal de Minas Gerais / The main objective of the present work was the study of PGA of B. megaterium production process and purification. The following production aspects were investigated in flasks and/or fermenter: the study of the purity of the culture, the standardization and the conservation of the inoculum, the optimization of the total time of cultivation (germination/propagation and production), the volume of the inoculum, the nutritional requirements of the B. megaterium for the production of the enzyme, the pH, the concentration of dissolved oxygen and the nutrients feeding mode. It was concluded that the conservation of the microorganism in cryovials, in presence the glycerol, showed to be efficient. The germination took 11 hours and the production, 24 hours, which is enough time for the process to reach the maximum production of the enzyme. With respect to the inoculum volume, 3 mL of inoculum/75 mL of medium (108 espores/mL of inoculum), in flasks of 500 mL, were found to be the appropriate conditions for its production. Different sources of carbon and nitrogen were studied and it was verified that glucose, glycerol and concentrations of amino acids above 10.0 g/L repress the synthesis of the enzyme. The largest production of PGA for B. megaterium occurs when the microorganism grows consuming free amino acids, phenylacetic acid (inductor), in the presence of some factor present in the cheese whey. The different components of the cheese whey were investigated and it was verified that none of the macronutrients is responsible for the significant effect of the whey on the production of the enzyme. This work allowed an increase on the enzymatic activities from 56 UI/L and 8,6 UI/g cell, obtained in the first experimental run with the standard inoculum, to 220 UI/L and 65 UI/g cell with the following conditions: 10 g/L of amino acids, 19,6 g/L of cheese whey, 0,4 g/L of salts, 2,7 g/L phenylacetic acid (AFA), initial pH 8,0, absence of pH control, addition of AFA in the beginning of the cultivation and control of dissolved oxygen around 20% of the saturation. During a research stage in Portugal, an investigation on the PGA production by Escherichia coli was also carried out in order to study the technique of adsorption in expanded bed and comparison of the conditions of production of the enzyme for E. coli with those used for B. megaterium. The maximum PGA production using E. coli was 400 UI/L. The same operational conditions used for E. coli were tested for B. megaterium and they didn't result in the enzyme production. In the study of the concentration and purification of the enzyme produced by B. megaterium, the ethanol precipitation technique was optimized. In addition, the ultrafiltration and adsorption in expanded bed (EBA) techniques were also studied. Using ultrafiltration, at 4ºC, enzyme concentrations up to 1541UI/L was reached, without thermal inactivation. The optimization of the previously study conditions results in: flow rate of 0.03 mL/s for the initial 22 mL of ethanol and 0.25 mL/s for the 98 mL of remaining ethanol, with adjustment of the pH of the broth for 6, without the previous dialysis. In the application of the adsorption in expanded bed the dynamic binding capacity found for PGA in clarified broth was about 5 UI / mL of adsorbent, with recovery of 30% of the initial enzyme. A similar value for the dynamic binding capacity was found for PGA of E. coli. / Este trabalho teve como objetivo principal estudar o processo de produção e purificação de PGA de B. megaterium. A produção foi estudada em frascos agitados e/ou fermentador, abordando-se os seguintes aspectos: estudo da pureza da cultura, padronização e conservação do inóculo, otimização do tempo total de cultivo (germinação/propagação e produção), volume do inóculo, requerimentos nutricionais do B. megaterium para a produção da enzima, pH, concentração de oxigênio dissolvido e modo de alimentação. A partir desses ensaios concluiu-se que a conservação do microrganismo em criotubos, na presença de glicerol, mostrou ser eficiente. Tempo de germinação de 11 horas e 24 horas na etapa de produção são os suficientes para atingir máxima produção da enzima. Quanto ao volume de inóculo, 3 mL de inóculo/75 mL de meio (108 esporos/mL de inóculo), em frascos de 500 mL, foram as condições ótimas encontradas para produção deste. Diferentes fontes de carbono e nitrogênio foram estudadas e verificou-se que glicose, glicerol e concentrações de aminoácidos acima de 10,0 g/L reprimem a síntese da enzima e que a maior produção de PGA por B. megaterium ocorre quando o microrganismo cresce consumindo aminoácidos livres, ácido fenil acético (indutor), na presença de algum fator presente no soro de queijo. Os diferentes componentes do soro de queijo foram investigados e verificou-se que nenhum dos macronutrientes é o responsável pelo significativo efeito do soro na produção da enzima. Este trabalho permitiu o aumento das atividades enzimáticas de 56 UI/L e 8,6 UI/g célula, obtidas no primeiro ensaio realizado com o inóculo padronizado, para 220 UI/L e 65 UI/g célula com as seguintes condições: 10 g/L de aminoácidos, 19,6 g/L de soro de queijo, 0,4 g/L de sais, 2,7 g/L de ácido fenil acético (AFA), pH inicial 8,0, ausência de controle de pH, adição de AFA no início do cultivo e controle de oxigênio dissolvido próximo de 20% da saturação. Foi também realizado, durante estágio em Portugal, estudo de produção de PGA por Escherichia coli, visando estudo posterior da técnica de adsorção em leito expandido e comparação das condições de produção da enzima por E. coli com as utilizadas para B. megaterium. A máxima produção de enzima obtida foi de 400 UI/L. As mesmas condições operacionais utilizadas para E. coli foram testadas para B. megaterium e não resultaram na produção de enzima. No estudo da concentração e purificação da enzima produzida por B. megaterium foi otimizada a técnica de precipitação com etanol, cujo estudo se iniciou em trabalho prévio, e estudadas as técnicas de ultrafiltração e adsorção em leito expandido (A.L.E). A ultrafiltração conseguiu concentrar a enzima até atividade final 1541UI/L. A temperatura mostrou ser uma variável importante, conseguindo-se evitar inativação térmica da enzima concentrada realizando-se a técnica a 4ºC. No processo de precipitação com etanol, os melhores resultados foram obtidos com vazão de 0,03 mL/s para os 22 mL iniciais de etanol e de 0,25 mL/s para os 98 mL de etanol restantes, com ajuste do pH do caldo para 6, sem a realização prévia de diálise. Na aplicação da adsorção em leito expandido a capacidade de ligação dinâmica encontrada para a PGA em caldo clarificado foi próxima de 5 UI/ mL de gel, com recuperação global de 30 % da enzima inicial. Valor similar para a capacidade de ligação dinâmica foi encontrado para PGA de E. coli.

Made available in DSpace on 2016-06-02T19:56:40Z (GMT). No. of bitstreams: 1 3185.pdf: 3522652 bytes, checksum: 063df10d4f80507d55f3980b84be9243 (MD5) Previous issue date: 2010-03-31 / Universidade Federal de Sao Carlos / Penicillin Acylases are enzymes of great industrial importance, being used mainly for production of 6-aminopenicillanic acid, 6-APA from penicillin G. In the late 90's, its use for synthesis of β-lactam semisynthetic antibiotics began on an industrial scale, an alternative in the context of "green chemistry" to the chemical synthesis of high environmental impact. The aiming thesis was to evaluate the influence of the growth media on the strains of Bacillus megaterium that will be used in the cloning of the gene pac in own B. megaterium, aiming at a larger number of copies of the gene, thus increasing the levels of expression of the enzyme penicillin G Acylase (PGA). It was determined the main metabolites produced by strains ATCC 14945 QB 1551 and PV 361 (the latter given by Professor Patricia Vary, corresponding to the wild QB 1551 with all megaplasmids deleted, which will be cloned with enzyme PGA). Evaluated medium has some influence on the metabolism of the organism, in order to optimize the production of the enzyme in cultures of high cell concentrations. The analysis of experimental data showed the strain PV 361 reached the highest cell concentration 16,6 g / L (dry weight), in fed-batch cultivation with the SNB medium with cheese whey (10,0 g / L), compared with 12,5 g / L without the presence of whey and 9,6 g / L in the LB-B medium. In cultures in SNB medium with whey the concentration of lactic acid reached 22,1 g/L, compared with 13,2 g/L in the absence of whey. However for the LB-B medium, the main product was acetic acid with a concentration of 6,7 g /L. Kinetic parameters were calculated as μmax, Yx/s, during the fed-batch cultivations in a bioreactor in stirred tank and aerated in a bench scale (5 L and 2L). The kinetic model for growth and production of metabolites was defined from the test phase in bioreactor batch. The operation of fed-batch culture and pH control were not effective for improving the production of biomass, because it had a high lactic acid production (up to 8 g / L in phase with the batch means SNB) and acetic acid (above 6 g / L at the end of the batch with LB medium-B), concentrations that led to the inhibition of growth of the microorganism and induced its sporulation, biological phenomenon that once started is difficult to reverse and that hinders the production in high cell density (HCDC). / Penicilinas acilases são enzimas de grande importância industrial, sendo utilizadas principalmente para produção de ácido 6 - aminopenicilânico, 6-APA, a partir de penicilina G. Em fins da década de 90, seu uso para síntese de antibióticos β- lactâmicos semi-sintéticos teve início em nível industrial, como uma alternativa no âmbito da química verde , aos processos químicos de síntese, de alto impacto ambiental. Este mestrado teve por objetivo avaliar meios de crescimento celular para linhagens de Bacillus megaterium que foram usadas na clonagem de seu gene pac no próprio B. megaterium, visando a um maior número de cópias do gene, aumentando assim os níveis de expressão da enzima penicilina G acilase (PGA). Foram determinados os principais metabólitos produzidos pelas linhagens ATCC 14945, QB 1551 e PV 361 (este último cedido pela Profa. Patrícia Vary, correspondendo à linhagem selvagem QB 1551 com todos os megaplasmídeos deletados, na qual será clonada a enzima PGA). Avaliou-se a influência do meio sobre o metabolismo do microrganismo, com o objetivo de otimizar a produção da enzima em cultivos de altas concentrações celulares. A análise dos dados experimentais permitiu identificar que o meio no qual a linhagem PV 361 atingiu a maior concentração celular em cultivos em batelada alimentada foi o SNB com soro de queijo (10,0 g/L ), alcançando concentração celular de 16,6 g/L (massa seca), comparado com os 12,5 g/L sem a presença do soro e 9,60 g/L no meio LB-B. Em cultivos no meio SNB com soro, a concentração de ácido lático atingiu de 22,1 g/L, comparado com 13,2 g/L na ausência do soro. Já para o meio LB-B o produto principal foi o ácido acético com uma concentração de 6,7 g/L. Os parâmetros cinéticos ajustados foram: μmax = 0,285 h-1 e 0,106 h-1 com e sem o soro respectivamente, obtidos utilizando o algoritmo simulated annealing (SA) junto com o algoritmo de Levenberg-Marquardt (LM). Foram calculados parâmetros cinéticos como μmax, Yx/s, na fase de batelada alimentada em cultivos em biorreator tipo tanque agitado e aerado, em escala de bancada (2L e 5 L). O modelo cinético de crescimento e de produção de metabólitos foi definido a partir de ensaios em biorreator na fase batelada. A operação do cultivo em batelada alimentada e o controle de pH não se mostraram efetivos para melhorar a produção da biomassa, pois se teve uma alta produção de ácido lático (acima de 8 g/L na fase batelada com o meio SNB) e acético (acima de 6 g/L ao final da batelada com meio LB-B), concentrações que levaram à inibição do crescimento do microrganismo e induziram sua esporulação, fenômeno biológico que uma vez inciado é de difícil reversão, e que dificulta ainda mas a produção em alta densidade.

Bacillus subtilis is a gram positive soil bacterium that is surrounded by many other microorganisms its environment. That is why it is necessary for the bacterium to be able to fight with these microorganisms for the nutrients and living space. B. subtilis contains the modules in its genetic make-up that improve its ability to compete. These modules are called the toxin-antitoxin systems. This Diploma Thesis is trying to identify yet undescribed extracellular toxins produced by the wild type BSB1 strain of B. subtilis. The related microorganism Bacillus megaterium was used as a competing bacterium. The contact-dependent or independent manner of killing the competing bacterium was demonstrated using this model. By deletion analysis and comparisons of the genomes of the various strains of B. subtilis, the SPβ prophage was first identified as a region containing an unknown toxin(s). Analysis of the extracellular proteome of B. subtilis subsequently revealed an unknown toxin (or toxin complex, respectively) of the molecular weight exceeding 100 kDa. Even more fascinating was the finding that such a large protein molecule is resistant to the pancreatic protease, trypsin. Subsequent non-enzymatic cyanogen bromide cleavage of the extracellular proteins and their analysis by mass spectrometry revealed...

Předložená disertační práce se zabývá studiem produkce a degradace polymerních materiálů s využitím mikroorganismů. Hlavní pozornost je upřena ke studiu produkce polyesterů bakteriálního původu - polyhydroxyalkanoátů. Tyto materiály jsou akumulovány celou řadou bakterií jako zásobní zdroj uhlíku, energie a redukční síly. Díky svým mechanickým vlastnostem, kterými silně připomínají tradiční syntetické polymery jako jsou polyetylén nebo polypropylén, a také díky své snadné odbouratelnosti v přírodním prostředí, jsou polyhydroxyalkanoáty považovány za ekologickou alternativu k tradičním plastům vyráběným z ropy. Polyhydroxyalkanoáty mají potenciál najít řadu aplikací v průmyslu, zemědělství ale také v medicíně. Významná část předložené práce je zaměřena na produkci polyhydroxyalkanoátů z odpadních substrátů pocházejících především z potravinářských výrob. Testována byla odpadní syrovátka nebo odpadní oleje z různých zdrojů. Právě využití levných odpadních substrátů je strategií, která by mohla přispět ke snížení ceny polyhydroxyalkanoátů a tím usnadnit jejich masové rozšíření. Podle výsledků dosažených v této práci jsou právě odpadní olejové substráty velice perspektivní cestou k ekonomicky rentabilní biotechnologické produkci polyhydroxyalkanoátů. Další část předložené práce se zabývá studiu spojení metabolické role polyhydroxyalkanoátů a stresové odpovědi bakterií. V této práci bylo zjištěno, že expozice bakteriální kultury řízené dávce etanolu nebo peroxidu vodíku významně navýší dosažené výtěžky a to o přibližně 30 %. Po aplikaci výše zmíněných stresových faktorů došlo k aktivaci metabolických drah vedoucí k odbourání stresového faktoru z média. Výsledkem bylo navýšení poměru NAD(P)H/NAD(P)+, což vedlo k částečné inhibici Krebsova cyklu a naopak aktivaci biosyntetické dráhy polyhydroxyalkanoátů. Mimoto došlo k významnému navýšení molekulové hmotnosti výsledných materiálů. Podle těchto výsledků se regulovaná aplikace vhodně zvolených stresových podmínek zdá být zajímavou strategií, která vede nejen k navýšení celkových výtěžků, ale také významnému zlepšení vlastností polymeru. Poslední část disertační práce se zabývala studiem procesu biodegradace polyuretanových materiálů. Polyuretanové eleastomery byly modifikovány rozličnými biopolymery za účelem navýšení jejich biodegradability. Tyto materiály byly posléze vystaveny působení směsné termofilní kultury jako modelového systému, který simuluje přirozené konsorcium bakterií. Přítomnost testovaných materiálů v kultivačním médiu vedla k neobvyklým růstovým charakteristikám bakteriální kultury. V průběhu prvních několika dní byl růst kultury silně inhibován, nicméně po překonání této neobvykle dlouhé lag-fáze došlo k intenzivnímu nárůstu kultury. Hlavní podíl na hmotnostním úbytku testovaných materiálů během experimentů měl samovolný rozpad materiálů, nicméně byl pozorován i vliv bakteriální kultury, kdy míra biotické degradace závisela na použitém modifikačním činidle. Nejvyšší míra biotické degradace byla pozorována u polyuretanového materiálu modifikovaného acetylovanou celulózou. Lag-fáze byla způsobena uvolněním nezreagovaného katalyzátoru (dibutylcínlaurát) a polyolu do kultivačního média. Bakteriální kultura se však po čase dokázala na přítomnost toxických látek v médiu adaptovat nebo je dokázala eliminovat.

<p>Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. </p><p>The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. </p><p>Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.</p>